Regulatory mechanisms controlling store-operated calcium entry

Calcium influx through plasma membrane ion channels is crucial for many events in cellular physiology. Cell surface stimuli lead to the production of inositol 1,4,5-trisphosphate (IP3), which binds to IP3 receptors (IP3R) in the endoplasmic reticulum (ER) to release calcium pools from the ER lumen. This leads to the depletion of ER calcium pools, which has been termed store depletion. Store depletion leads to the dissociation of calcium ions from the EF-hand motif of the ER calcium sensor Stromal Interaction Molecule 1 (STIM1). This leads to a conformational change in STIM1, which helps it to interact with the plasma membrane (PM) at ER:PM junctions. At these ER:PM junctions, STIM1 binds to and activates a calcium channel known as Orai1 to form calcium-release activated calcium (CRAC) channels. Activation of Orai1 leads to calcium influx, known as store-operated calcium entry (SOCE). In addition to Orai1 and STIM1, the homologs of Orai1 and STIM1, such as Orai2/3 and STIM2, also play a crucial role in calcium homeostasis. The influx of calcium through the Orai channel activates a calcium current that has been termed the CRAC current. CRAC channels form multimers and cluster together in large macromolecular assemblies termed “puncta”. How CRAC channels form puncta has been contentious since their discovery. In this review, we will outline the history of SOCE, the molecular players involved in this process, as well as the models that have been proposed to explain this critical mechanism in cellular physiology.


Introduction:
Calcium is a crucial secondary messenger that serves a multitude of functions ranging from subcellular signaling to organ system-level changes. It serves many roles in biological processes, including growth, disease, and death [1][2][3]. The levels of calcium in the cytosol are maintained ~100nM by many ways, including calcium pumps and exchangers on the plasma membrane and various organelles. The extracellular calcium concentration is 1-1.5 mM [1]. The calcium ion gradient across the plasma membrane (PM) is maintained by calcium efflux pumps and ion channels, in addition to calcium reuptake mechanisms in ER. Sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps use an active transport mechanism to move calcium across the concentration gradient from the cytosol into ER lumen [4,5]. Upon cellular stimulation by agonist actions at plasma membrane receptors, a multitude of signaling cascades starts that lead to the production of inositol 1,4,5-trisphosphate (IP3) due to phospholipase-Cmediated cleavage of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) [6]. Once produced, IP3 binds to and activates the IP3 receptors (IP3Rs) on the ER membrane to promote calcium release from the ER lumen into the cytosol. This decrease in calcium levels in the ER leads to intracellular calcium store depletion [7]. Store depletion leads to the activation of an ER transmembrane protein known as stromal interaction molecule 1 (STIM1), which then binds to Orai1 channels on the plasma membrane to promote store-operated calcium entry (SOCE) [7,8]. The channel formed by Orai1 and STIM1 is known as the calcium release-activated calcium (CRAC) channel. A general overview of SOCE is presented in Figure 1.
Store-operated calcium entry is a predominant mechanism for calcium entry from extracellular milieu to increase cytosolic calcium after store-depletion. It also serves to refill the ER calcium stores after IP3-mediated calcium release [9,10]. The most important role SOCE plays is by a sustained calcium entry that can last from several seconds to up to an hour in some cellular systems [11,12]. One specific case where this differential role of calcium entry plays a crucial role is the activation of T cells. For example, for activation of nuclear factor of activated T cells (NFAT), which regulates IL-2 production, a sustained calcium entry is needed which lasts for several minutes [13]. While for activation of NFkB, calcium oscillations of lower frequency from Orai1 channels is required. Finally, CaMKII, a calcium sensitive protein in the cells needs a calcium current of higher frequency for its activation. Briefly put, cellular functions mediated by calcium involve both frequency and amplitude modulation [14]. Calcium entry from the plasma membrane is essential for shaping the spatiotemporal aspects of calcium transients, including the amplitude and duration of calcium release events. Calcium entry serves many functions such as secretion, gene transcription, enzyme activation, and other functions.
Efficient SOCE requires the formation of multiple Orai1-STIM1 complexes at defined ER:PM junctions known as "puncta" [15][16][17][18]. A key difference between CRAC channels and other calcium channels such as voltage/ligand gated ion channels is their ability to conduct calcium currents at a negative membrane potential, and without ligand binding [19]. This independence of CRAC channels from extraneous factors makes SOCE ideal for mediating the role of calcium in the cells. Taken together, the ability of CRAC channels to selectively conduct calcium influxes to mediate many cellular functions helps maintain the spatial and temporal regulation of cellular signaling.
In this review, we look at the historical origins of SOCE and recap a few landmark studies that established the characteristics of SOCE. We will delve into the proteins that form the CRAC channel complex, such as Orai1 and STIM1, and how these proteins are regulated. In addition to STIM1, STIM2 is another member of ER resident calcium sensors that promote SOCE. Orai2 and Orai3 are genes in the Orai family with Orai1. Proteins outside these two families such as TRP channels also play a role in SOCE. Finally, we will also review the effect of SOCE associated regulatory factor (SARAF) and role it plays in SOCE. SARAF promotes calcium dependent inactivation, but how it functions as an inactivator of SOCE is not well known. Our review outlines the key modulators of SOCE, and more importantly, the modulators of SOCE that undergo S-acylation, and how this important post-translational modification regulates SOCE.

History of SOCE
The earliest known proposal for SOCE was introduced by the Putney group in 1985 as outlined by their model for receptor-regulated calcium entry, in which they propose the mechanism for sustained calcium entry from extracellular matrix upon agonist binding to extracellular receptors [20]. Termed as the capacitative calcium entry model, they carefully analyzed the calcium release from the ER and entry from extracellular milieu. A few years laters, Hoth and Penner identified similar inward rectifying calcium current in mast cells upon depletion of intracellular calcium stores using IP3 [21]. Zweifach and Lewis then showed store-depletion in T cells shows the similar calcium current upon depletion of ER calcium stores using thapsigargin, a SERCA pump inhibitor [22]. Later, studies published by Liou  IP3-mediated calcium release: Cleavage of membrane phospholipids by phospholipase C (PLC) leads to production of IP3 and diacylglycerol [6]. Liberated IP3 binds to IP3 receptor calcium channels on ER membranes, and leads to calcium efflux from the ER lumen [29]. Depending on the strength and duration of agonist stimulation, IP3R activation leads to decrease of calcium levels in the ER lumen which is termed as store-depletion [7,29]. The role of IP3 in activating calcium release from endoplasmic reticulum was initially discovered by experiments by the Berridge group in blowfly salivary glands based on the ability of those tissues to respond to hydrolysis of PIP2 [30]. Subsequently, the role of IP3 as a secondary messenger was to mobilize internal calcium stores in mammalian cells was demonstrated in saponin-permeabilized hepatocytes [31]. Application of IP3 led to a brief rise in intracellular calcium levels followed by a sustained calcium plateau [31]. Capacitative calcium entry was the first model to explain the calcium entry observed in cells upon emptying intracellular calcium pools [20]. This idea originated from the observation that calcium entry from the extracellular milieu lasted for a long duration after the levels of IP3 returned to the baseline. These observations were made in rat parotid gland upon carbachol [32] application and rabbit ear artery upon application of noradrenaline [33]. Based on the biphasic nature of agonist-induced increase in the cytosolic calcium, he proposed the initial increase is a result of calcium release from the ER due to the action of IP3 followed by influx of calcium through channels in the plasma membrane until the levels of calcium in the ER reaches to a significant level that stops the entry [20].

CRAC channels:
In an effort to accurately define the mechanism of CRAC currents and to distinguish it from other calcium currents, capacitive calcium entry was later re-named to store-operated calcium entry (SOCE). CRAC channels conduct calcium currents at a negative membrane potential. The voltage independence of CRAC currents was first observed in patch recordings conducted on Jurkat T cells treated with various cell mitogens such as PHA, a substance known to activate Tcell signaling pathway [19]. A similar current was also observed in these cells with low extracellular calcium. In a separate set of experiments conducted in mast cells, intracellular dialysis with IP3 and extracellular application of substance P generated a similar low-noise current (1-2pA) in patch-clamp recordings [34,35]. This current developed in these cells with a concomitant increase of intracellular calcium. The currents observed by both these groups showed similar features such as inward rectifying current-voltage relationship, voltageindependent gating, very high calcium selectivity, an extremely low unitary conductance, and extracellular calcium-dependent feedback inhibition.  [21]. The crucial experiments conducted by Lewis and colleagues using perforated-patch clamp and thapsigargin in T cells showed the similarity between TCR mediated calcium currents and CRAC currents [22,43,44]. These studies led to definitive proof that CRAC currents are mediated by intracellular ER calcium stores.
The research into how ER calcium depletion leads to CRAC channel activation led to three main hypotheses: 1) diffusion of an activating factor released from the ER to the plasma membrane, 2) targeting of active CRAC channels to the PM by membrane fusion, and 3) conformational coupling between ER calcium sensor and PM calcium channel. Here we will discuss these proposals in detail.

Calcium influx factor (CIF):
The earliest proposal for a diffusible mediator that is released from the intracellular organelles into the cytosol and extracellular milieu that can activate calcium influx into the cells was presented in early 1990s. Application of phytohaemagglutinin (PHA)treated Jurkat T cell supernatant to P388D1 macrophage cells showed sustained but fluctuating calcium increase. A diluted version of the supernatant decreased the amplitude and increased the latency of the calcium flux [45]. Interestingly, NG115-401L cells did not show CIF-induced calcium release [46]. In later studies it was shown that NG115-401L cells do not express endogenous STIM1 [47]. Treatment of the extract with alkaline phosphatase neutralized the effect of these extracts. These observations led to a proposal where application of receptor agonists leads to production of a diffusible factor that activates extracellular influx of calcium [7,8]. These models have now been disproved after extensive research into the molecular players involved in the formation of CRAC channels. Of the three models highlighted above, the conformation coupling model came close to explaining the mechanism of SOCE as we know now. But it involves IP3R channel as the calcium sensing protein, which we now know as STIM1 and STIM2 proteins. We now know that Orai proteins form the calcium channels and STIM proteins function as ER calcium sensors. These proteins form oligomers at ER:PM junctions where they promote CRAC channel formation and SOCE.

TRP channels
Transient receptor potential (TRP) channels are ion channels that show diverse ion selectivity, activation mechanisms, and physiological functions. All TRP channels share some common features such as six transmembrane domains, their selective permeability toward cations, and remarkable sequence similarity. The discovery of TRP channels directly resulted from the studies focused toward understanding Drosophila visual transduction in the laboratory of Craig Montell [53,54]. The earliest research into TRP channels as SOCs was conducted owing to the knowledge that the PLC-mediated calcium release, and subsequent calcium entry is crucial for phototransduction in drosophila. The drosophila trp and trpl mutants showed a significant decrease in light-induced calcium influx and a transient response to light [55,56]. This information, in conjunction with the knowledge that PLC is crucial for fly vision, formed a hypothesis that TRP channels are PLC-operated calcium channels. Cloning and characterization of these channels proved this idea [57,58]. The protein encoded by the trp gene localizes to the eye, contains four N-terminal ankyrin repeats, and importantly, a topology similar to voltage gated ion channels. Whole cells currents recorded from Sf9 insect cell expression system showed the channels encoded by trp gene are activated upon ER calcium store-depletion, and are moderately selective for calcium than sodium (PCa:PNa ~ 10:1) [59]. subfamily of TRP channels are found in invertebrates [65] and zebrafish [66]. Some distantly related TRP channels are also found in fungi falling into the TRPY subfamily [67,68].
In Drosophila, three TRPC members are expressed in the eye that play an important role in phototransduction. TRPL and TRPγ, in addition to TRP, are the three proteins work in concert for successful operation of fly vision [69,70]. TRPL and TRPγ have ~50% sequence identity with TRP in the six TM domains, and only differ in the TRP domain, which contains highly

Mechanism of Drosophila TRP activation and function:
The Drosophila trp can be activated in tissue culture setting by blocking the SERCA (sarcoplasmic endoplasmic reticulum calcium ATPase) pumps that maintain the calcium gradient between ER lumen and cytosol [59,69].
Thapsigargin irreversibly blocks SERCA pumps and cause ER calcium leak, which will then activates store-operated calcium channels [36,74]. However, in vivo observations do not support the in vitro analyses. Thapsigargin-mediated SERCA blockade or IP3 receptor activation does not promote calcium influx or affect phototransduction [75][76][77]. Some studies also evaluated the potential effect of diacylglycerol, another product of PLC hydrolysis of PIP2, on calcium influx and phototransduction. Isolated photoreceptor cells from drosophila wild type and rdgA mutants (these mutants have inactive DAG kinase) show constitutive TRP activity.
Recently, endocannabinoids produced in Drosophila photoreceptor cells in response to light have been shown to activate TRP channels [78]. In addition, using flies engineered to express genetically encoded ER calcium indicator, the sodium/calcium exchanger has been shown to result in rapid calcium release from the ER upon light exposure [79].
Mammalian TRP channels: The seven TRPC proteins in mammals are divided into four groups based on sequence homology [80,81]. Similar to Drosophila TRPC proteins, the mammalian counterparts also include three/four ankyrin repeats, 6 TM domains, and high sequence homology in the TRP box domain in the N-terminus [82][83][84][85][86]. Similar to Drosophila TRPCs the activation of mammalian TRP channels can be activated in cultured cells through PLC activation [85,87]. The specific mediator in the PLC pathway that ultimately activates these channels is still unknown, and the current hypotheses include activation by IP3, DAG, and the calcium released from the internal stores. TRPC1-4 proteins have been shown to be activated upon store-depletion, either by IP3 or thapsigargin treatment in cultured cells expressing these proteins as they lead to calcium influx [85,87,88]. Calcium influx factor was proposed to be a factor to activate these channels to promote calcium influx [45,46,89]. Additional hypotheses also included conformational coupling between IP3R and the TRP channels [51]. Some models also suggested internalization of TRPC1/3/4 channels upon store depletion [90,91]. Other TRPC proteins, such as TRP6/7 are activated by diacylglycerol, and show similar results like that of Drosophila TRP channels, including the uncertainty of whether the mode of activation of these channels is directly by DAG [90,92].
Another complexity in mammalian TRPC channel activation comes from the specific activators of these variants. For example, mouse TRPC2 in vomeronasal neurons has been shown to be activated by DAG, whereas the same protein in mouse sperm has been shown to be activated by calcium release [93,94]. Whether this differential activation is due to alternate splicing or different multimerization is unknown [95,96]. TRPC3 also shows similar differential activation between DAG and store-depletion. Some reports also suggest direct binding between TRPC3 and IP3R as a mode of activation of these channels [87,92,97]. This diversity in activation mechanisms between different TRP channels makes the role of TRP channels more compelling to study.

TRP Channels and their role in SOCE:
Of the different roles TRP channels play, the crucial one is store-operated calcium entry [98]. TRP channels have low calcium selectivity, which distinguishes them from conventional CRAC channels or voltage-gated calcium channels. As such, TRP channels are high conductance and non-selective cation channels with varying permeability ratios between calcium and sodium [99,100]. The mechanism of TRP channel activation and gating ranges from changes in cytosolic calcium concentration, to membrane depolarization, and external cellular stimuli [21]. The loop between transmembrane domains 5 and 6 form the channel pore of these TRP channels. Four individual TRP subunits form an active channel complex to promote calcium entry [101,102]. The domain architecture of different TRP family proteins regulates the functions of these proteins. For example, TRPC, TRPV, and TRPA channels have repeats of ankyrin regions in their N-terminus [102] and TRPC and TRPM channels have a conserved TRP domain adjacent to the last transmembrane domain [53]. TRPM subfamily of TRP channels have a catalytic kinase domain in their C-terminus.
These proteins also have a highly conserved TRP box sequence (Glu-Trp-Lys-Phe-Ala-Arg) and proline-rich sequence that regulates signal transduction and gating [103]. In addition, coiled-coil domains in C-and N-terminals aid in multimerization in some TRP channel subfamilies [104].
These coiled-coil domains also regulate the channel binding to STIM1, the ER calcium sensor, to regulate SOCE [105]. A C-terminal calmodulin-and IP3R binding region in TRPC channels is known to regulate both store-independent, and store-dependent calcium entry [106,107].
Prior to the discovery of Orai1/STIM1 complex, TRP channels were believed to be the reason behind calcium entry observed in mast cells upon IP3/ionomycin stimulation [21]. Mammalian TRP channels also play a huge role in cation influx in non-excitable cells, despite being nonspecific to calcium and sodium. However, since the discovery of STIM1 as the ER calcium sensor and Orai1 as the pore forming unit of CRAC channels, the role of TRP channels in conventional SOCE has shifted [15,23,26,108,109]. Further research into the role of TRP channels in SOCE in the recent studies provide more accurate insight into their role in this process.
Different subfamilies of TRP channels are expressed in different cell types and carry out specific functions in only these cell types. For example, TRPC channels are expressed in endothelial cells, where they mediate cell shape, blood vessel tone and permeability, angiogenesis, and leukocyte trafficking by modulating calcium entry in these cells [110][111][112][113][114]. TRPC4 knockout mice show impaired vasorelaxation of the aortic rings and no IP3 and BAPTA-induced calcium influx in aortic endothelial cells from these animals [115]. TRPC subfamily proteins are also expressed in other non-excitable cells where they regulate SOCE such as platelets, pancreatic beta cells, salivary gland cells [116,117]. Another important process where SOCE plays an important role is in fertilization of the mammalian egg. A signaling cascade is activated following fusion of the sperm with the egg the results in activation of several GPCR signaling mediators finally resulting in store-operated calcium influx. A putative isoform of TRPC2 helps in the calcium influx in the egg cell that is mediated by the glycoprotein ZP3 in mice [94].
Several TRPC proteins has been found in brain and tissues in the central nervous system [118,119]. In rodents, expression of TRPC2 is involved in the pheromone detection response [96,120]. In mammalian brains, TRPC3 is activated following brain derived nerve growth factor (BDNF) activation of receptor tyrosine kinase TrkB [121]. This pathway is known to aid in neuronal differentiation, synaptic plasticity, and neurotransmitter release. TRP channels play roles across animal kingdom in sensory physiology such a visual transduction in Drosophila [122], olfaction, mechanosensation, and osmosensation in C. elegans [123], acid, heat, and pain responses in mice [124]. Support for TRP channels acting as store-operated channels came from the studies done using TRPC1 expression in vitro which showed increased calcium entry after store-depletion [87,88,125]. One key difference in these experiments was even though these cells showed calcium entry upon store-depletion, this current was different from the biophysical properties observed in CRAC currents (ICRAC) [88,126,127]. Experiments later determined that TRPC1 is assembled with the Orai1-STIM1 complex [128,129]. Immunofluorescence and confocal microscopy in human salivary gland cells showed colocalization of Orai1, STIM1, and TRPC1 at the plasma membrane upon store-depletion [128]. This is an interesting observation considering the information available to us now regarding the role of STIM1 and Orai1 in SOCE, and how it differs from calcium entry observed in response TRPC channels activation. Further research into the binding of TRPC1 with STIM1/Orai1 added some clarity to this observation. Orai1 gating is regulated by STIM1 activation and dimerization upon store-depletion, where STIM1 translocates to the ER:PM domains and binds to Orai1 at STIM1-Orai1 activation region (SOAR) and the C-and N-termini of STIM1 [130,131]. Activation and gating of TRPC1 is mediated by negatively charged aspartate residues which bind STIM1 at both the SOAR domain as the polybasic domain [132].
Finding an explanation to how and if the calcium entry observed from Orai1:STIM1 complex upon store-depletion differs from TRPC1:STIM1 complex led to many interesting hypotheses.
One such hypothesis is that the local increase of cytosolic calcium near ER:PM junction upon store-depletion activates cytosolic TRPC1 channels, which then gets embedded into the plasma membrane to be activated by STIM1 [11,133]. This model can explain the spatio-temporal differences observed in terms calcium oscillations between ICRAC observed in Orai1:STIM1 complexes and ISOC observed in TRPC1:STIM1 complexes. These differences also affect the physiological and pathological outcomes of cytosolic calcium rises in cells. One specific example is the activation of nuclear factor of activated T-cells (NFAT) vs activation of NF-kB.
Calcium entry observed Orai1 upon T-cell stimulation leads to activation of NFAT [134][135][136], while TRPC1-dependent calcium entry leads to activation of NFkB [137]. Interestingly, knockdown of Orai1 in Jurkat cells inhibits both SOC and CRAC currents with no effect upon knockdown of TRPC1 or TRPC3 [138]. In addition, calcium entry observed from the Orai1:STIM1:TRPC1 complex leads to insulin release from pancreatic beta cells [139,140], platelet activation during blood clotting, and SNARE complex formation for adipocyte differentiation and adiponectin secretion [141]. Furthermore, calcium entry from this complex contributes to prostate and colon cancer cell migration [142,143]. Finally, calcium entry observed in STIM1-Orai1-TRPC1-TRPC4 complexes plays a pivotal role in right ventricular hypertrophy [144].

Discovery and cloning of Orai1 and STIM1:
Feske and colleagues reported abnormalities in T-cell activation in infants born to consanguineous parents that manifested as severe combined immunodeficiency (SCID) [25].
They found increased CD4+ T cell counts and inability of these T cells to produce IL-2 upon stimulation with phorbol 12-myristate 13-acetate (PMA), concanavalin A, and anti-CD3 stimulation. Exogenous application of IL-2 restored the proliferative deficiencies of these cells.

Further analyses into DNA binding of transcription factors showed lack of NFAT interaction with
the DNA with no difference in AP-1, NFkB, and Octamer binding proteins. They also found no differences in expression of NFAT, but the dephosphorylation and nuclear translocation was affected in these T cells [145]. In subsequent experiments using PMA+ionomycin and increasing concentrations of extracellular calcium, they found that higher levels of extracellular calcium rescued IL-2 production in these T cells [146]. They concluded that dysregulation in upstream signaling events play a role in NFAT binding to DNA elements and ultimately result in SCID [25].
During this time, it was discovered that there exists two different calcium mobilization patterns in T cells after stimulation. An immediate transient calcium spike [147] followed by a sustained calcium influx from extracellular milieu [20,148]. The transient calcium release is capable of activating downstream proteins such as NFkB and JNK, but NFAT activation needs sustained elevations to promote dephosphorylation by the calcium-dependent phosphatase calcineurin [149][150][151][152]. Calcium imaging of T cells obtained from the patients with SCID showed dysregulated calcium entry upon stimulation with anti-CD3, ionomycin, and thapsigargin. More importantly, membrane hyperpolarization with valinomycin did not alter calcium entry abnormalities found in these cells, suggesting the lack of calcium entry is not a result of depolarization [153]. These intriguing results warranted further research into the calcium entry in T cells upon their activation and the ion channels that promote this sustained calcium entry.
Development of RNAi screening as a research tool in early 2000s opened avenues for high throughput identification of proteins that mediate specific signaling pathways [154,155]. This tool helped answer the question that persisted in the field of calcium entry about the ion channel that is activated upon store depletion. Even though TRP channels were proposed to be the CRAC channels, the results from the research did not support these hypotheses. The very low unitary conductance and high calcium selectivity observed in CRAC currents were not displayed by these TRP channels. Two important siRNA screens performed at this time identified the proteins involved in this influx, and more importantly pinpointed the possible mechanism how these proteins function [27,156]. These two screens were performed in the Drosophila S2 cell line and were screened for calcium entry upon store-depletion. Before these screens, patch clamp whole-cell recordings done in these cells showed store-operated calcium entry in these cells that had the same biophysical properties as CRAC channels in mammalian cells [157].
The siRNA screen performed by Feske and colleagues in Drosophila S2 cells identified known proteins that affect calcium influx in addition to NFAT regulatory proteins [27]. When they used this RNAi screen to identify potential regulators of Ca +2 /calcineurin mediated NFAT activation, they found a kinase that negatively regulated exogenously expressed NFAT. In addition, they also found other candidates in this screen that alter calcium levels in the cytosol such as SERCA, Homer, and Stim. Around the same time, Zhang and colleagues independently performed a genome-wide RNAi screen to identify the components of CRAC channels in Drosophila S2 cells. Probing for hits that resulted in an inhibition of calcium influx after storedepletion by thapsigargin, they identified 11 transmembrane proteins including Stim. Of note was another four transmembrane protein olf186-F, which showed reduction in SOCE and CRAC currents. They followed up this experiment with an overexpression paradigm, which showed a three-fold increase in CRAC currents, which was further increased to eight-fold upon coexpression with STIM [156]. These two pivotal experiments identified Orai1 and STIM1 as the proteins that form the CRAC channel.
At the same time these RNAi experiments were being conducted, Feske and colleagues continued their research into understand the T-cell activation abnormalities in the SCID infants.
Using whole-cell patch-clamp electrophysiology performed on T cells from control and SCID patients, they showed that T cells obtained from some SCID patients lacked SOCE.
Furthermore, they also found that exogenous expression of STIM1 in these SCID cells did not rescue SOCE in these cells [135]. Next, they used a combined approach of Drosophila RNAi screen and linkage analysis with single-nucleotide polymorphism array to screen for regulators of SOCE and nuclear import of NFAT. T cells obtained from SCID infants and relatives of these infants were analyzed for SOCE deficits and SNP mapping arrays using their genomic DNA.
This in conjunction with Drosophila RNAi screen for NFAT regulators both pointed to a gene olf186-f in Drosophila and to the TMEM142A gene on chromosome 12 in humans which they named Orai1 [158]. The genomic DNA sequencing showed a mutation of an arginine to tryptophan at 91 residue (R91W) on Orai1 as a direct result of SNP from cytosine to thymine.
Finally, exogenous expression of wild-type Orai1 in these T cells from SCID patients restored SOCE with the currents showing a biophysical footprint consistent with CRAC currents.
STIM1 is mammalian homolog of Drosophila Stim, which is essential for CRAC channel activation in human T cells. Roos and colleagues showed, in Drosophila S2 and human HEK293 and Jurkat cell lines that STIM1 is highly conserved and silencing of STIM1 abrogates SOCE [159]. Following this finding, Zhang and colleagues expressed the EF-hand motif in Jurkat T cells and analyzed SOCE patterns in these cells. Along with wild-type EF-hand motif, they also used STIM1 1A3A and 12Q mutants that show constitutive activation based on activation mechanics of EF-hand motifs [160,161]. These findings were also independently confirmed by another group using siRNA-based silencing of STIM proteins in HeLa cells [23]. These experiments showed that STIM1, upon calcium store-depletion, gets activated and moves to ER:PM sites [24]. This discovery along with the discovery of Orai1 as CRAC channel pore potentiated a barrage of research into these proteins in SOCE.
The discovery of Orai1 as pore-forming subunit of CRAC channels was a direct result of the RNAi approaches in Drosophila S2 cells by several groups as outline above. Following the research into calcium deficits observed in SCID human patients, two different groups pursued RNAi screens focused toward decreases in NFAT translocation into nucleus, and they individually found the same four transmembrane plasma membrane protein with intracellular C and N termini olf186-F[26, 153], and named it CRACM1 [26]. The human homolog of this protein was eventually named Orai1. There are three homologs of Orai proteins with high sequence identity, with up to 90% identity in the transmembrane regions.
The experiments using the T cells obtained the human SCID patients increased our understanding of the role of Orai1 in SOCE. Genomic sequencing from DNA obtained from healthy and SCID patients in their family led to discovery of a missense mutation in the Orai1 gene (R91W). This mutant channel lacked SOCE, which could be restored by transformation of the T cells from those patients with wild-type Orai1 [158]. These observations were strengthened by co-expression of Orai1 with STIM1 in HEK293 cells that generated CRAC currents about a hundred times larger than endogenous CRAC currents observed in the untransfected cells [162][163][164]. Further, experiments done in yeast using human Orai1 and STIM1 expression system showed that these two proteins are sufficient to a calcium influx that recapitulates CRAC currents [165]. Another important observation from these set of experiments is the stoichiometry of Orai1:STIM1 (1:2 ratio) in SOCE. However, the precise stoichiometry of the Orai1 and STIM1 proteins in the CRAC puncta remains unanswered [166].
Overexpression of Orai1 and STIM1 in HEK293 cells helped identify the pore forming subunit of CRAC channels. The definitive proof for Orai1 as the pore forming subunit came from experiments that showed the importance of acidic residues in the transmembrane domains of Orai1. Residues E106 and E190 are two important glutamic acid residues that line the extracellular pore of Orai1 channel. Mutation of these residues to aspartate and glutamine respectively decreased the calcium influx, increased monovalent cation current, and made the channel cesium permeable [167]. These experiments strongly supported Orai1 as the pore forming unit of CRAC channel.

Orai1/STIM1 clustering/puncta
Orai1 in the plasma membrane and STIM1 in the ER membrane are diffusely distributed in resting conditions in cells with replete ER calcium stores. The Orai1-STIM1 complexes come to close proximity near the ER:PM junctions where they redistribute into multiple CRAC channel clusters to promote efficient calcium entry upon store depletion [16]. These clusters were denoted as "puncta" upon observation under fluorescence microscopy of tagged-STIM1 [18,23,24,109,168,169]. These ER:PM junctions are regions within the cell where PM and ER are held in close apposition to each other within ~10-20nm where SOCE occurs [18,170]. Upon store-depletion, STIM1 accumulates near the thin cortical tubules of the ER [170]. An interesting observation in CRAC puncta formation is the longevity of these puncta. Depending on the downstream effect of calcium influx, the puncta can be active for a few minutes or up to an hour.
How these puncta can stay active to promote calcium entry for this duration is still not completely understood. Even more compelling is the ability of STIM1 to translocate to the sites of already formed puncta repeatedly upon subsequent store-depletion [171]. A multitude of factors regulate this behavior to increase the number of cortical ER tubules near the plasma membrane. One mechanism is a secretion-like coupling model which includes redistribution of F-actin into cortical layers [172]. STIM1 has been known to interact with microtube attachment protein EB1 and maintain ER tubule length [173]. STIM1 also binds to plasma membrane using its polybasic domain which might strengthen the STIM1 localization in the puncta [16]. The SOAR region of STIM1 also has a cholesterol binding domain which has been shown to bind to cholesterol rich regions in the plasma membrane (27459950). Several additional proteins like extended synaptogamins and septins maintain the integrity of these ER:PM junctions [174,175].
Cytosolic calcium elevation led to increased translocation of E-Syt1 to ER:PM junctions, which subsequently recruited a phosphatidylinositol transfer protein (PITP) Nir2 to these junctions to strengthen ER:PM junction stability [175]. E-Syt2 and E-Syt3 also regulate the ER:PM junctions, albeit without any regulation through cytosolic calcium [176]. Interestingly, siRNA mediated knockdown of E-Syt proteins decreased the number of ER:PM contact sites but did not affect SOCE [176]. This explains the possibility that E-Syt proteins maintain the stability of ER:PM junctions with no specific effect on Orai1 or STIM1. Another protein that has shown a role in regulating SOCE as well as long-term maintenance of ER:PM junctions is an ER transmembrane protein TMEM110, also known as junctate [177]. Junctate is a calcium binding protein in the ER membrane that forms supramolecular complexes with IP3 receptors and TRPC3 calcium channels. Junctate has an EF-hand domain in its ER luminal side, which upon mutation led to impairment of calcium binding as well as CRAC channel activation independently of store-depletion [178,179]. In addition to these proteins mentioned above, many other proteins play a crucial role in maintenance of stable ER:PM junctions that help CRAC channel formation and function.

Clinical implications of mutations in Orai1 and STIM1:
Mutations observed in Orai1 and STIM1 lead to several diseases in humans, and the phenotype is broadly based upon whether the mutations are loss-of-function or gain-of-function. The lossof-function mutations show immunological deficits such as immunodeficiency, ectodermal dysplasia autoimmunity, muscular hypotonia, and defects in tooth formation. The gain-offunction mutations show constitutive CRAC channel activity and abnormal cytosolic calcium levels which lead to diseases affecting musculoskeletal system. Gain-of-function mutations lead to the disorders tubular aggregate myopathy (TAM), York platelet syndrome, Stormorken syndrome, thrombocytopenia, and bleeding disorders. The common mutations involved in CRAC channelopathies are discussed in table 1 below.

Mechanism of activation of SOCE:
The discovery of STIM1 as ER calcium sensor that activates SOCE upon store-depletion was a pivotal moment in furthering the field of store-operated calcium entry and led to discovery of Orai1 and its role in CRAC channel formation [23]. Soon after the discovery of STIM1, multiple research groups discovered a four transmembrane cell surface protein with cytosolic C-and Ntermini in Drosophila and its homologs in mammalian cells [26,156,158]. Overexpression of this protein in T cells isolated from patients with SCID restored the calcium entry deficits in those cells. In addition, co-expression of Orai1 with STIM1 in HEK293 cells showed calcium currents in these cells that matched the biophysical properties of CRAC currents [163]. This observation settled the debate on the constituents of CRAC channels and what proceeded was decadeslong effort into understanding the mechanism of CRAC channel formation, how these two proteins convalesce at the ER:PM junctions, and the driving forces behind this process. The CRAC channel formation is interesting in its mechanism in that two proteins that exist in two different subcellular compartments choreograph the signaling to form the active channel to promote SOCE at distinct ER/PM junctions [24,128,130,165,180]. In this next section, we will discuss the specific research that highlighted the mechanism of activation of CRAC channel components.

STIM1
Depletion of ER calcium stores initiates a cascade of events start with activation of STIM1 and culminates with calcium entry from the extracellular milieu. STIM1 is a dimer of two single-pass type I ER transmembrane proteins. In the N-terminus, there is a canonical and a noncanonical EF hand motif that play a role in calcium sensing in the ER [181,182]. Immediately adjacent to the EF hand motifs, there is a sterile alpha motif (SAM) domain. On the cytosolic side, there is a calcium-activating domain (CAD) which is also known as STIM-Orai activation region (SOAR) which binds to the Orai1 channel [131]. The cytosolic C-terminus also contains three coiled-coil domains (CC1, CC2, CC3) that help maintaining STIM1 in its inactive folded conformation.
Toward the end of the C-terminus, there is a polybasic domain which binds to the PIP2 in the plasma membrane [131]. In addition to these domains, STIM1 also has and inhibitory domain (ID), a proline-serine rich domain (P/S domain), EB1 binding domain (EB) in its cytosolic side.
These domain act in conjunction with each other upon store-depletion to colocalize with Orai1 to form CRAC channels.
The calcium binding EF hand motifs are required for STIM1 to sense ER calcium store levels.
This was confirmed using experiments with mutations in the EF hand motif. The mutations D76A, D78A, and E87A reduce the affinity of this motif to bind to calcium and show constitutive puncta formation as well as CRAC currents in resting cells [23,24,163]. The EF-SAM domains of STIM1 and STIM2 show similar affinities to calcium in vitro, however, structural differences in the two proteins results in dramatically different abilities to oligomerize in response to store depletion [181,183].
The first step in the CRAC channel activation after ER calcium store-depletion is the conformational change in STIM1. This was elucidated in experiments conducted using cytosolic fragment composed of amino acids 233-685, which is capable of activating SOCE regardless of store-depletion [184]. The most important characteristic of CRAC channels is that the calcium flux occurs at the ER:PM junctions where Orai1 and STIM1 colocalize and bind to each other [109]. This local calcium flux is crucial for both ER calcium refilling as well as the calcium influx needed for cellular signaling. Further experiments involving truncation mutants of STIM1 led to identification of STIM1-Orai1 activating region (SOAR/CAD), the stretch of amino acids that can activate SOCE without store-depletion. It is, however, interesting to note the difference in the amino acid sequences identified by individual groups ranged from 342-448 [185], 344-442 [131], and 339-444 [186]. Despite these differences, the consensus is that the SOAR/CAD region can activate CRAC currents independently of store-depletion. Further research into CAD domain also showed that CAD binds both C and N termini of Orai1, but the strength of binding is higher at the C terminus [185]. This binding between Orai1 and CAD is mediated by coiled-coil interplay between CC2 region of STIM1-CAD and the anti-parallel polar residues in the C terminus of Orai1 [187].
In resting conditions, STIM1 is diffusely localized throughout the ER, but redistributes into clusters that are termed "puncta" [185]. In resting conditions, STIM1 is in a compact conformation that keeps it from interacting with Orai1. Upon store-depletion, a conformational change in STIM1 leads to an extended conformation with its N-terminus reaching toward the plasma membrane where it binds to the C-terminus of Orai1 [18,24,168]. Following storedepletion, a complex choreography of intramolecular events happens between several domains of STIM1 that stabilizes STIM1 in its extended conformation to promote binding with Orai1 and calcium entry. The ER:PM sites of cells are held in close apposition to promote STIM1 and Orai1 binding to promote CRAC channel formation by a multitude of accessory proteins such a septins, extended synptogamin proteins (ESyts), junctate, and others [18]. The extended conformation of STIM1 was proposed to trap Orai1 into puncta leading to channel gating to promote SOCE [188,189]. STIM1 exists as a dimer in cells with calcium replete stores in the resting state. This was confirmed using the C-terminal cytosolic fractions in vitro which formed dimers in solution. The specific domains involved in dimerization of STIM1 were further resolved using coimmunoprecipitation and fluorescence photobleaching of individual fragments [190]. The coiledcoiled domain fragments and CAD domain independently can form dimers in vitro. The CAD domain dimerization is interesting because STIM1 binding to Orai1 is also mediated by this domain. The CC1 domains can form dimers but are weak and unstable [190,191]. This strengthens the arguments that STIM1 dimerization is a complex process with multiple domains binding to each other after store depletion.
The CAD domain of STIM1 can independently and constitutively activate Orai1 and promote CRAC currents. Based on a crystal structure of CAD domain, hydrophobic residues form hydrogen bonds between two monomers stabilizes the dimer [186]. In addition, interactions between the CC2 and CC3 regions of the STIM1 are purported to stabilize the extended conformation of STIM1. One interesting finding in the structure of CAD domain is the role of the CC2 and CC3 helices in binding to and activating Orai1 channels. In resting inactive state, the two CC2 helices (in a dimer) are in a parallel configuration in a tight hairpin structure with CC3 helices. Upon store-depletion, these CC2 helices pivot and twist to an antiparallel orientation, allowing CC3 to extend out and allow for binding with Orai1 [108,187,192,193]. These are highly complex and precise molecular movements in a restricted space between ER and PM within seconds of store-depletion.
The EF hand domains of STIM1 residing in the ER lumen also undergo dimerization upon release of calcium from their binding pockets. In resting state, these domains exist as compact monomers bound to calcium [181]. NMR-resolved calcium-bound STIM1/2 EF-SAM fragments show an α-helical structure with a canonical (cEF) and non-canonical (nEF) EF hand domains.
The cEF domain contains a helix-loop-helix structure that binds to calcium and nEF domain does not bind to calcium but stabilizes the cEF through hydrogen bonding [194]. Upon calcium release, the EF-SAM domains unfold leading to conformational changes in luminal and cytosolic domains of STIM1. Mutations in the glutamate/phenylalanine/lysine residues in the EF-hand domain led to puncta formation irrespective of calcium depletion [192]. This led to a proposal that calcium release from EF-hands leads to STIM1 oligomerization upon store-depletion. Livecell imaging and FRET studies using STIM1 mutants added credence to this hypothesis, as they show increased FRET between STIM1 fused with YFP and CFP in RBL cells, which was reverse upon calcium addback [16]. Furthermore, FRET experiments conducted using Orai1-CFP and STIM1-YFP also show a spatiotemporal correlation between STIM1 oligomerization and Orai1-STIM1 binding [195].
Taken together, how calcium depletion in the ER leads to activation of STIM1 shows a complicated picture of multiple events happening in a precise sequential order mediated by several domains of STIM1. First, the binding of calcium ions to the EF-hand keeps the EF-SAM in a compact state which helps interactions between CC1 and CAD regions of STIM1 in the cytosolic side. An inhibitory clamp formed by intramolecular interactions between CC1/2/3 regions of STIM1 helps keep it in inactive state [168,193,196]. Store-depletion and calcium dissociation from EF-hands leads to a conformational change that releases this clamp and formation of a coiled-coil dimer between CC1 domains. Experiments done using isolated cytosolic STIM1 fragments show that in resting state, these cytosolic fragments are tightly bound which are extended upon binding to Orai1 [197]. Mutations in the CC1 regions that cause spontaneous activation of STIM1 also show a similar phenotype [108]. Finally, several leucine residues (L251, L261, L419, and L416) in the CAD region play a crucial role in extension of STIM1 toward plasma membrane for STIM1 binding to Orai1 [108,198,199]. This intra-andintermolecular choreography of STIM1 proteins upon store-depletion controls SOCE. An overview of important binding events between STIM1 and plasma membrane lipids is shown in Figure 2.

Orai1
There are three homologs of Orai proteins (Orai1, Orai2, Orai3) in humans, and all three proteins calcium entry upon store-depletion with varying biophysical properties. They can form both homo-and hetero-multimers. Orai1 and Orai3 also assemble as heteromultimers to promote store-independent calcium channels that are regulated by arachidonic acid or leukotriene C4 [200][201][202][203]. These are called ARC channels and LRC channels respectively.
Orai protein subunits are composed of approximately 300 amino acids. Early evidence from coimmunoprecipitation and FRET experiments suggested that these proteins are oligomers in functional CRAC channels [195,[204][205][206]. Experiments using preassembled tandem Orai1 multimers and coexpression with dominant-negative Orai1 mutants showed that Orai1 may form homotetramers in CRAC channels similar to other ion channels [207]. Using photo-bleaching of individual fluorophores in tandem Orai1-STIM1 multimers suggested a similar result of four Orai1 molecules with two STIM1 dimers, which was confirmed using FRET [208]. These results were replicated in live mammalian cells as well as cellular lysates obtained from lymphocytes [17,209]. However, the X-ray crystallographic structure of Drosophila Orai revealed a hexameric stoichiometry challenging these observations [210].

Channel gating and biophysical properties of Orai1 channel
X-ray crystallographic structure helped resolve the oligomer status of Orai1, and more importantly it helped us understand the structure of Orai1 channel pore and the role the subunits play in channel function. This structure shows Orai1 as a four transmembrane protein with cytosolic C and N termini. The TM1 forms the channel pore with the Orai1 N terminus. TM2 and TM3 shield the pore from the membrane and TM4 and C terminus extend away from the channel pore. The channel pore is 55 A long the extracellular side of which form the channel opening. The channel opening is made of glutamate residues on TM1 (E178 is Drosophila, E106 in humans) that form a highly negative electrostatic region which also serves as a selectivity filter. The side chains of these glutamate residues extend into the central pore where the oxygen atoms of the carboxylic groups are in close proximity (~6 A). Below the selectivity filter is a region of hydrophobic amino acids followed by positively charged residues that extend into the cytosolic lumen. One interesting observation from the crystal structure is that the C terminal tails of Orai1 helices form anti-parallel helices with each other in the hexamer. These interactions are held together by hydrophobic interactions between leucine residues at 316 and 219 (273 and 276 in humans). Mutations at these residues, such a L273S/D and L276D lower the coiled-coil probability of the C terminus as well as inhibit STIM1 binding and channel activity [205,211,212]. However, an NMR structure between Orai1 272-292 fragment and 312-387 STIM1 fragment shows the anti-parrel orientation doesn't change upon binding, but only leads to a small change in the angle or Orai1 helices [187]. The information obtained from these multitude of experimental approaches gave a picture, despite a hazy one, of how CRAC channels are gated, especially how the allosteric modulators regulate Orai1 channel function.
This was eventually clarified in a cryo-EM structure published recently [213].
It has been shown that store-depletion leads to slow activation of Orai1 channels (seconds to tens of seconds) as a result of increased probability for channels to an open state [214]. Based on these data, a non-linear gating mechanism was proposed to regulate CRAC channel gating where these channels exhibit a 'modal gating' mechanism where the channels alternate between silent and high open probability [215]. Modal gating mechanism was based on an observation that 2-APB, a non-competitive antagonist of IP3 receptor widely used as ICRAC inhibitor, elicits strong SOCE at low concentrations and inhibition at high concentrations. This hypothesis proposed that STIM1 binding to the Orai1 channel increases the time spent by these channels in the high open probability state. And because the frequency of these transitions is lower, the calcium entry through these is enhanced.
The research into identifying the location of CRAC channel gate has taken a considerable amount of time owing due to lack of crystal structures. Four seminal structural studies helped us elucidate the crystal structure of this Orai1 channel [210,213,216]. The identification of drosophila Orai gave the first clues into the gating mechanism for these channels. The glutamate residues in the extracellular side of the transmembrane (E106, E178 in Drosophila), as explained earlier in the review, were hypothesized to form a selectivity filter by selectively binding calcium over other divalent cations. Mutation of a valine at 102 (V174 in Drosophila) in the TM1 domain to alanine or serine made the channels constitutively active [217]. The dOrai structure also shows the hydrophobic side chains of valine residues making extensive connections protruding into the pore in the transmembrane that presents a de-solvation barrier for ions in closed configuration [210,218]. Using Terbium (Tb3+) luminescence and disulfide crosslinking, others have also shown that STIM1 binding to Orai1 leads to a conformational change in the extracellular side near E106 and V102, and that this short segment forms a STIM1-dependent barrier [219]. Based on these observations, V102 was proposed as a hydrophobic gate, which was eventually refuted and F171 has been shown to be the hydrophobic gate (as discussed below).
Surprisingly, mutation of glycine and arginine residues in the intracellular side of TM1 region of Orai1 at 98 and 91 respectively resulted in constitutive activation of Orai1 channels. This led to a speculation whether there is another gate in the in the cytosolic side of the channel [220].
Based on these observations, R91 was thought to be the physical gate at the intracellular side that leads to dilation of the helices upon STIM1 binding. G98 was suggested to serve as a hinge, upon which the N terminus can rotate to allow for calcium entry. In addition, a phenyl alanine at 99 is on the opposite side of the pore helix to G98. STIM1 binding has been proposed to evoke a conformational change that exposes G98 while concealing F99 away from the channel pore [221]. The Drosophila Orai crystal structure revealed basic residues in the immediate inner cytosolic end of channel pore which led to another hypothesis where these residues stabilize the closed channel through either binding of anions or electrostatic repulsion of cations near this pore [210]. With the knowledge obtained from structural and functional studies using Orai1 and STIM1 mutants, several hypotheses have been put forward to explain the mechanism of activation of Orai1 by STIM1. STIM1 has been proposed to activate Orai1 in stepwise manner, first by initial binding to the C terminus for docking and a subsequent weaker binding to the N terminus to initiate conformational changes in the TM pore leading to pore opening [222].
A stretch of conserved acidic amino acids of Orai1 have been shown to confer calcium selectivity. These include residues at E106, E190, D110, D112, and D114 all of which are either in the TM1, TM3 or TM1-2 loop. Mutation aspartate residues to alanine resulted in increased cesium permeability in these channels. In addition, mutation of E106 residue in the putative channel pore resulted in the same increase in cesium permeability. These mutants also increase in pore diameter as evidenced by increased permeability of methylammonia derivatives. Finally, E106D mutant also increases the fast calcium dependent inactivation time latency compared to wild-type Orai1.
The X-ray crystal structure of open conformation of Orai1 was resolved at 3.3 A using fiducialassisted cryo-electron microscopy [213]. The hydrophobic amino acids within the channel pore, F99 and V102 (F171 and V174 in human), as mentioned earlier in the review were thought to function as channel gate [210]. This structure shows the channel pore lined by acidic residues facing the extracellular entrance by D184, D182, Q180 and E178 followed by polar residues V174, F171, and L167 lining the middle of the pore, and positively charged K163 and K159 lining the pore on the intracellular side. In addition, TM1-TM2 subunits from each monomer of Orai1 form an inverted V shaped turret about the selectivity filter formed by E178 on the extracellular side [213]. E178, Q180, D182, and D184 amino acids that line the extracellular side make up the negative electrostatic surface to attract cations near the extracellular pore.

Selectivity and permeation of CRAC channels
One of the most distinguishing properties of CRAC channels is their selectivity toward calcium over other cations. CRAC channels show 1,000 times higher selectivity over sodium.
Mutagenesis of residues lining the channel pore provided insights into the remarkable selectivity of Orai1 channels toward calcium. Alanine substitutions of E106, D110, D112, D114 residues in the TM1 region and TM1-TM2 loop and E190 in the TM3 region all result in loss of calcium selectivity and increased cesium permeating. In addition, these mutations also diminish calcium dependent inactivation of these channels. Cesium permeability analyses on these channels show that the loss of selectivity is due to increase in the minimal diameter of these channels [223]. E190Q mutation in the TM3 region also resulted in loss of calcium selectivity [204] [167].
Also, alanine substitution of D180 residue changed these channels from calcium selective and inward rectifying to sodium/cesium selective and outward rectifying [28]. These observations highlight the selectivity filter in the Orai1 channels is formed by the conserved acidic residues lining the outer pore of the channel.
Cysteine scanning experiments performed on these channels showed that the channel pore is narrow lined by the TM1 region leading to low permeability of large cations and low unitary conductance. Lack of cysteine reactivity suggests that the E190 residue doesn't form the channel pore. In addition, cysteine substitution of aspartate residues at 110, 112, 114 in the TM1-TM2 loop did not change calcium selectivity of these channels. [165,224]. These results show that TM1 lines the channel pore and each E106 residue from each monomer (effectively six E106 residues in a Orai1 hexamer) forms the selectivity filter in the extracellular side of Orai1 channel.
Taken together, the above results highlight the highly complex molecular choreography happening in the Orai1 and STIM1 proteins upon store-depletion which results in the highly calcium selective inwardly rectifying unitary currents.

SARAF
SARAF (SOCE-associated regulatory factor) is an ER transmembrane protein known to be bind to STIM1, keeping it in an inactive state. It primarily localizes to the ER membrane and possibly to the plasma membrane [225,226]. SARAF was discovered in a functional expression screen to identify proteins that affect mitochondrial calcium homeostasis [225]. HEK293 cells transfected with SARAF cDNA showed lower baseline cytosolic, ER, and mitochondrial calcium levels. In addition, siRNA-based knockdown of SARAF resulted in an increase of basal cytosolic and ER calcium levels. This suggested a role for SARAF in cellular calcium homeostasis.
Further experiments conducted using SARAF in conjunction with Orai1 and STIM1 led to deciphering its role in calcium entry. Electrophysiology recordings of Jurkat T cells and HEK293 cells showed SARAF regulates SOCE, specifically the slow calcium-dependent inactivation of CRAC channels [225]. Subsequent experiments conducted in SH-SY5Y and NG115-401L cell lines also showed that SARAF is expressed in the plasma membrane where it interacts with and negatively regulates ARC channels [227]. In addition, SARAF also plays a negative role in calcium entry mediated by TRPC1 channels [228].
How SARAF affects STIM1 is still not completely understood. Several reports indicate that SARAF interacts with STIM1 in resting conditions to keep STIM1 in its inactive state. Coimmunoprecipitation studies have shown that SARAF binds to STIM1, and this binding is mediated by the C-terminus of SARAF. More importantly, the TM and ER-luminal domains are not required for this interaction [225]. Interestingly, constitutively active STIM1 which has four glutamates mutated to alanine in its SOAR domain does not bind to SARAF and has deficits in slow calcium-dependent inactivation [229]. The physical interaction between STIM1 and SARAF has been confirmed by multiple reports. In addition, SARAF has been shown to bind Orai1 and EFHB (EF hand containing family member B) [229][230][231][232][233].
The nature of SARAF binding to STIM1 and Orai1 is interesting, especially when it is put into SOCE context. SARAF binds to STIM1 in resting conditions to keep it from spontaneously activating. Upon store-depletion, co-immunoprecipitation experiments have shown that SARAF immediately dissociates from STIM1 within the first minute, but these two re-bind to each other following the dissociation [230][231][232]. It is possible that SARAF binds to Orai1 after storedepletion, not STIM1. The exact sequence of events by which SARAF binds/releases STIM1 and Orai1 during SOCE is difficult to determine using existing techniques. Experiments conducted using fragments of these proteins show that SARAF interacts with STIM1 at the CC1 and CTID domains of STIM1 [229]. TIRF microscopy using STIM1-mCherry and SARAF-GFP show that these two proteins co-localize at the ER:PM junctions upon store-depletion using thapsigargin. Co-immunoprecipitation of the SARAF C and N termini with STIM1 shows that SARAF binds to STIM1 with its C terminus [225].
Membrane phospholipids play a crucial role in the interaction between SARAF and STIM1/Orai1. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a membrane phospholipid that mediates many functions in cellular signaling. It has been shown that the CRAC complex translocates between PI(4,5)P2 poor and rich regions during SOCE, and this PM localization regulates SOCE. STIM1 interaction with the C terminus of Orai1, and STIM1 polybasic domain interaction with the plasma membrane, are both dependent on SARAF and PI(4,5)P2 [230]. In addition, these PI(4,5)P2 microdomains need to be tethered by stabilizing proteins such as extended synaptogamin 1 (E-Syt1) and septin4 [230]. The temporal resolution of interactions of SARAF and STIM1 with membrane phospholipids still needs to be properly evaluated to accurately determine if these phospholipids modulate CRAC channel trafficking to PM microdomains. More recently, the SOAR domain of STIM1 has been shown to bind interact with plasma membrane phospholipids. Interactions between lysine residues (382, 384, 385, 386) in the SOAR domain with plasma membrane phospholipids PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI (3,4,5)P2 are crucial for the stability of ER:PM junctions as well as SOCE.
How SARAF regulates slow calcium-dependent inactivation is still unknown. Multiple hypotheses have been proposed to explain this behavior. One of them is that an additional calcium-binding protein mediates SARAF action in slow calcium-dependent inactivation [234]. Calcium binding proteins have been known to modulate SOCE, and some of them have been discussed earlier in the review such as EFHB and calmodulin [235]. Other hypotheses include modulation by calcium sensing of STIM1 and Orai1.

GTP-dependent puncta formation
Based on the observation in live cells that STIM1 translocates to the plasma membrane from the ER membrane and an earlier hypothesis that STIM1 is transported to PM via a secretory pathway [236], puncta formation was hypothesized to be GTP-dependent. Mitochondria are known to accumulate near the ER:PM junctions and regulate calcium homeostasis [12,237]. In addition, depletion of intracellular GTP led to decreased calcium entry in rat lymphocytes which added weight to the argument that CRAC channel formation and puncta formation is GTP dependent [238]. Consistent with this observation, GTP depletion led to translocation of STIM1 to puncta. However, this required depletion of PI(4,5)P2 in addition to GTP depletion [239]. In addition, the GTP is depleted in these cells using oligomycin which inhibits ATP synthase but doesn't affect cellular ATP levels.

Microtube-associated STIM1 translocation
As discussed earlier in the review, STIM1 binds to the EB1 protein that is known to modulate microtubule growth. In B cells, treatment with the anti-mitotic agent nocodazole, which inhibits polymerization of microtubules, did not show the puncta formation observed in untreated cells [169]. Pull down assay with EB1, EB2, and EB3 proteins showed STIM1 binds to EB1. This was also confirmed using immunocytochemistry and co-immunoprecipitation experiments [173]. In addition, treatment of cells with ML-9, a myosin light chain kinase inhibitor, led to reversal of CRAC puncta as well inhibition of SOCE [171]. These observations led to a hypothesis that STIM1 traffics to the ER:PM junctions by microtubule-assisted transport mediated by its binding to EB1. A major drawback to this hypothesis is that neither EB1 nor microtubule extension directly or indirectly regulate SOCE. In addition, this model shows that STIM1 and Orai1 proteins are confined at the junctions after store-depletion but does not offer any explanation to why this happens.

Phosphatidylinositol-mediated membrane sorting
Early experiments conducted on STIM proteins led to discovery of a C-terminal domain rich with basic residues that interact with PM phospholipids, such as PI(4,5)P2 [240,241]. This polybasic domain was also found to mediate the inward rectification of SOCE currents [131]. HeLa cells treated with wortmannin and LY294002, inhibitors of phosphatidyl inositol 3-kinase (PI3K) and PI4K respectively, led to inhibition of STIM1 puncta formation as well as SOCE [242]. In addition, the binding between STIM1 and Orai1 was differentially modulated by the levels of PI(4,5)P2 [241]. Decreased PI(4,5)P2 concentration in the membrane ordered regions showed reduced thapsigargin-mediated Orai1-STIM1 binding, and this affinity was reversed in membrane disordered regions [242]. This led a hypothesis that membrane phospholipids play a role in CRAC channel formation and SOCE. However, depletion of phosphoinositides in cells overexpressing Orai1 did not affect either STIM1 puncta formation or SOCE [242]. In addition, these experiments showing role of phosphoinositides was shown using inhibitors of membrane phospholipid concentrations that does not specifically distinguish if the result is due to the effect of the protein function or loss of membrane integrity.

Diffusion-trap model
The diffusion-trap model postulates that STIM1 undergoes a conformational change that exposes its C-terminal domains toward plasma membrane, where it binds to membrane phospholipids and stochastically binds to Orai1 channels laterally diffusing in the PM [188,189].
In agreement with this hypothesis, super-resolution microscopy experiments conducted using tagged Orai1 and STIM1 constructs showed that these proteins diffuse randomly in resting conditions. Upon store-depletion, these proteins slow down at distinct ER:PM junctions allowing them to accumulate and form puncta [188]. Additionally, single particle tracking analysis shows single STIM1 and Orai1 particles diffusing freely before getting trapped at the junctions. Deletion of the polybasic domain in the C-terminus of STIM1 did not show the decreased mobility observed in WT STIM1. These experiments also show that almost all STIM1 and Orai1 particles stay within the puncta [188,243].
This does not fully explain certain crucial elements of SOCE. The polybasic domain was hypothesized to act as a trap to attract Orai1 to STIM1. But mutant STIM1 devoid of this polybasic domain is capable of binding with Orai1. Orai1 binding by itself can also trap STIM1 within ER:PM junctions which raises the possibility if STIM1 is acting as the trap or Orai1 [242].
Interestingly, we have also found that STIM1 can form puncta in the absence of Orai1, but Orai1 cannot form puncta in the absence of STIM1 [244,245] . The strong binding between Orai1 and STIM1 in the puncta could also mean there is an equivalent amount of these protein particles enter and leave the puncta, thereby maintaining a dynamic equilibrium at these ER:PM junctions [188]. Finally, single particle tracking and polydispersity analyses conducted on Orai1 and STIM1 proteins show that the mobility of these proteins decreases after store-depletion, but these proteins are also confined in compartmentalized membrane regions before and after store-depletion [243]. Based on these observations, the binding of Orai1 and STIM1 upon storedepletion appears to be more complicated than a simple diffusion model.

S-Acylation of STIM1 and Orai1
Orai1 and STIM1 were shown to be accumulated in the lipid raft domains upon depletion of intracellular calcium stores and additionally depletion of these domains using methyl-betacyclodextrin impaired SOCE [246,247]. Early observations in our lab that a biphasic calcium release from ER is necessary for Fas-mediated apoptosis [248] led to a foray into delineating the molecular players involved in this calcium flux. Further analyses into this ER calcium release uncovered a role of PLC-y1-mediated IP3 production for calcium efflux via IP3Rs [248].
Concomitant experiments involving death receptor signaling revealed that dynamic S-acylation is a central component of TCR signaling. We found many TCR components such as Lck, Src, ZAP70, PLCy1 undergo S-acylation upon T cell activation [134,249,250]. This S-acylation was mediated by DHHC21 which was confirmed using siRNA mediated knockdown as well as a mutant mouse model known as depilated mice, which carry a functionally deficient DHHC21 with an in-frame deletion of phenylalanine 233 (F233). Noting that F233 is in a predicted calmodulin binding site, it was also shown that DHHC21 binds calmodulin and mediates differentiation of CD+ T cells into Th1, Th2, and Th17 lineages. Depilated mice also do not show phosphorylation of ZAP70 kinase, Src, PLCγ1, JNK, ERK1/2, and p38, the crucial regulators of T cell activation in response to TCR stimulation [251,252]. As a logical consequence, we hypothesized that Orai1 and STIM1 undergo S-acylation to regulate CRAC channel formation and function.
S-acylation is a reversible addition of lipid moieties to cysteine residues of target proteins that affects protein stability, function, conformation, and trafficking between compartments within a cell [253,254]. S-acylation is a post translational modification that is mediated by specific group of enzymes known as palmitoyl acyltransferases (PATs). PATs are also known as DHHC enzymes owing to the conserved aspartate-histidine-histidine-cysteine motif in their active site that carries out this reaction [253]. The different classes of DHHC enzymes distinguish among themselves by their selectivity toward substrates, lipid preferences, and mechanisms that activate and inactivate them [254]. Deficiencies in DHHC enzyme functions leads to a range of diseases ranging from cancers such as adenocarcinomas, lung cancer, bladder cancer, and breast cancer, to diseases that affect neurological functions such as epilepsy and schizophrenia, glioblastoma, and X-linked intellectual disability, and other systems [254][255][256][257][258].
Despite being discovered in early 1970s, the research in this area has not progressed owing to the difficulty of studying this important modification. The subsequent process of removal of lipid moieties from protein substrates, known as deacylation, is mediated by another set of enzymes known as acyl protein thioesterases (APTs) [259,260]. These enzymes act in concert to dynamically regulate protein function in a stimulus-dependent manner. How these DHHC and APT enzymes are activated and inactivated is still unclear.
DHHC enzyme were hypothesized to catalyze the addition of lipid moieties to proteins through a two-step ping-pong mechanism, where the cysteine residue in the active site of the enzyme receives the lipid group and then transfers it to the cysteine residues of target proteins [261].
This first step is known as auto-acylation which is followed by the transfer of acyl group to target proteins. This hypothesis was based on an observation of transient acyl enzyme intermediate in these DHHC enzymes. However, a close appraisal of the enzymatic process of DHHC enzymes do not show the characteristics of ping-pong mechanisms. Ping-pong mechanisms (also known as double-displacement reactions) usually involve modification of the enzyme followed by binding of two substrates in succession which results in addition a moiety to proteins [262,263].
The catalysis mediated by DHHC enzymes does not involve multiple substrates, and usually only involves one enzyme and one substrate. The ping-pong mechanism for DHHC enzyme function is a misnomer and the reaction mechanism is highlighted in Fig 3. The catalysis mediated by DHHC enzymes involves the uptake of acyl groups by the DHHC enzyme at the cysteine residue in its active site. This step, as explained earlier, is called auto acylation. The acyl group is then transferred to cytosolic cysteine residues of substrate, which leads to acylation of these substrate proteins. The enzyme is liberated after this transfer step, which helps the enzyme to return to the unacylated pool, where it can further S-acylate by the same process.
Following the hypothesis that Orai1 and STIM1 undergo S-acylation upon store-depletion, we found that treatment of Jurkat T cells with anti-CD3 or HEK293 cells with thapsigargin (an irreversible SERCA pump inhibitor that leads to ER calcium store-depletion) leads to stimulusdependent S-acylation of Orai1 and STIM1. The time kinetics of S-acylation followed the pattern observed in T-cell activation paradigms for both Orai1 and STIM1. Not surprisingly, the cysteinemutant versions of Orai1 and STIM1 that are incapable of undergoing S-acylation showed distinct calcium entry deficits as observed by Fura2 whole cell calcium imaging as well electrophysiological recordings. In addition, these mutant versions failed to colocalize upon store-depletion as observed by decreased puncta formation upon store-depletion. Finally, using an Orai1 construct fused to GCaMP6s, we were also able to show the role of Orai1 and STIM1 S-acylation in individual channels as evidenced by decreased GCaMP6s fluorescence [244,245].
The data obtained from the cysteine mutants help us understand the role of S-acylation in SOCE. Orai1 and STIM1 mutants that cannot undergo S-acylation show decreased calcium entry, but not complete abrogation of channel activity. One interesting observation between the cysteine mutants of Orai1 and STIM1 is that the Orai1 cysteine mutant (C143S) showed a drastic abrogation of channel function compared to Orai1 WT whereas STIM1 cysteine mutant (C437S) showed some calcium entry, albeit being non-significant. This might be explained by behavior observed using TIRF imaging of these mutants. These mutants still show some colocalization, but not the same degree observed in wild type proteins. This behavior can be explained as S-acylation is an additional mechanism that sequestrates these proteins into membrane subdomains such as lipid rafts where they undergo oligomerization to promote SOCE. The residue where STIM1 undergoes S-acylation is at the proximal end of its SOAR domain. As explained earlier in the review, the SOAR domain of STIM1 is crucial in the tethering of STIM1 to plasma membrane. The cholesterol binding domain in the SOAR region of STIM1 binds to cholesterol, which is mediated by isoleucine 364, and the S-acylation of STIM1 (C437) at a residue distal end of SOAR gives credence to the idea that S-acylation of STIM1 stabilizes the CRAC puncta at ER:PM junctions [264]. Many aspects of STIM1 binding to Orai1 at the plasma membrane such as the cholesterol binding domain binding to PIP2, the SOAR domain binding to the plasma membrane, and the role of polybasic domain in tethering STIM1 to the plasma membrane suggest S-acylation of STIM1 by a plasma membrane resident DHHC21. We believe a plasma membrane resident DHHC enzyme, probably DHHC21, S-acylates STIM1 and anchors its C-terminus in the plasma membrane, thereby stabilizing the CRAC complex at these ER:PM junctions to facilitate SOCE. These experiments suggest S-acylation as a governing mechanism for CRAC puncta formation [244,245]. An overview of the role of S-acylation of Orai1 and STIM1 in CRAC puncta formation is depicted in Figure 3.
At the same time as our research into S-acylation of Orai1 and STIM1, Demaurex group also showed that Orai1 undergoes S-acylation [265]. They also showed that S-acylation of Orai1 is critical for its recruitment to the immunological synapse as well as TCR-mediated calcium entry in Jurkat T cells. Using TIRF and confocal imaging techniques, they showed that S-acylation of Orai1 is critical for channel clustering as well as their trafficking to the lipid rafts. Finally, using overexpression analysis, they show that DHHC20 is the enzyme that mediates S-acylation of Orai1 in the plasma membrane. They also show DHHC3 and DHHC7 mediate S-acylation of Orai1 in the Golgi apparatus. These results align with our findings that Orai1 S-acylation is crucial for CRAC puncta formation and calcium entry.
One distinction between the findings of Demaurex group and ours is the enzyme that S-acylates Orai1 and STIM1 proteins [265]. Our hypothesis originates from observations in depilated mouse which has a functionally deficient DHHC21 whereas they show DHHC20 is the enzyme that S-acylates Orai1 in resting conditions. This is an interesting observation since their data does not include thapsigargin mediated ER calcium store-depletion or activation of T cells using CD3. In our experiments using depilated mouse (unpublished data) we observe DHHC21 also S-acylates Orai1. This can be explained by differential S-acylation of Orai1 by both DHHC20 and DHHC21, where DHHC20 S-acylates Orai1 in resting conditions, but upon store-depletion, DHHC21 increases this S-acylation and helps Orai1 translocate to lipid rafts where it can bind to STIM1. This stimulus-dependent activation of Orai1 and STIM1 by DHHC21 can also be explained by the structural features of this enzyme that DHHC20 lacks. DHHC21 has a predicted calmodulin binding motif in its C-terminus, and we found that DHHC21 can be activated by calcium (unpublished data). This suggests a mechanism where calcium released from ER upon IP3 binding to IP3R channels activates DHHC21 which in turn S-acylates Orai1 and STIM1. Consistently with this hypothesis, we have also found that Orai1 and STIM1 cannot undergo S-acylation in depilated mouse spleen and lymph nodes, which was also confirmed in HEK293 cells overexpressing DHHC21-Flag constructs with WT and deltaF233 (unpublished data).
S-acylation governing CRAC channel formation and SOCE adds another layer in regulating calcium signals in cell physiology. This secondary regulation of channel formation and function gives an additional way to fine tune calcium levels upon receptor activation to effectively process signaling events. Our results provide an alternative model for CRAC channel assembly and disassembly that is enzymatically regulated. This allows fine-tuning of SOCE and resultant spatio-temporal aspects of the calcium signal.

Conclusions:
Since the discovery of store-operated calcium entry, questions have lingered on how this important mechanism is regulated. Orai1 and STIM1 from spatially distinct subcellular localization fuse together near the ER:PM junctions to form CRAC channels and promote calcium entry [7,21,180]. The ER resident STIM1 and PM resident Orai1 move to these junctions independently of each other to facilitate the calcium entry [18]. As we outlined earlier, many hypotheses were proposed to explain this complicated process. All these hypotheses have fallen short in explaining crucial aspects observed in how these proteins could shuttle between membrane subdomains. We proposed and showed some evidence for S-acylation to play a role in the formation and CRAC channels. Recently, we also found new evidence for DHHC21, a PM-resident palmitoyl acyltransferase, to regulate SOCE.
Our hypothesis that S-acylation plays a role in store-operated calcium entry was derived from the inability of current models to explain the specific characteristics that make this process unique. As explained earlier in the review, Orai1 and STIM1 proteins show distinct features upon store-depletion, such as decreased mobility in the membrane, targeting to membrane subdomains, and dynamic nature of CRAC puncta, among other observations [169,188,243].
We based our hypothesis that S-acylation, being a process that is controlled by a set of enzymes, gives a dynamic control to Orai1 and STIM1 function. Figure 1: General overview of store-operated calcium entry (SOCE).    Agonist stimulation through the PLC-coupled receptors leads to IP3-mediated calcium release which leads to ER calcium store-depletion. As a result, calcium dissociates from the EFhand of STIM1 and leads to its activation. This leads to a conformation change in STIM1 which extends its C-terminus toward plasma membrane where it binds to Orai1 channels and form calcium-release activated calcium (CRAC) channels. CRAC channels promote calcium entry from extracellular milieu into the cells. One subunit of STIM1 dimer is shown here for simplicity. The CRAC channels are stabilized at the ER:PM junctions by a multitude of bindings between different domains of Orai1 and STIM1 with plasma membrane lipids upon store-depletion. Orai1 undergoes S-acylation at its C143 residue which shuttles the channels to lipid rafts. The SOAR domain of STIM1 binds to the N-terminus region of Orai1 and leads to activation of Orai1 channels. The cholesterol binding domain (CBD) in the SOAR domain also binds to the cholesterol rich phospholipids in the plasma membrane, which is mediated by I364 residue. The polybasic domain of STIM1 binds to the PI(4,5)P2 phospholipids in the plasma membrane which is mediated by the positively charged amino acids in the C-terminal tail of STIM1. Finally, STIM1 undergoes S-acylation at its C437 residue which is crucial for SOCE. One subunit of STIM1 dimer is shown here for simplicity.  S-acylation of Orai1 and STIM1 leads to CRAC puncta formation and stabilization of CRAC channels. Calcium dissociation from the EF-hand of STIM1 leads to conformation change in STIM1 which extends the C-terminal tail toward plasma membrane, where multiple domains of STIM1 interact with plasma membrane, including S-acylation of STIM1. Orai1 is then recruited to lipid rafts through its S-acylation. Together, STIM1 and Orai1 form CRAC channels.